Macrophage foam cells are major drivers of the development and pathology of atherosclerosis. Exposure of macrophages to sterol lipid and inflammatory TLR signals modifies gene regulatory networks controlling lipid homeostasis, which can favor foam cell formation. There exists a fundamental gap in our understanding of the molecular mechanisms by which TLR-lipid signal crosstalk impinges upon these networks and regulates foam cell formation. The long-term goal is to understand the molecular mechanisms governing foam cell formation, for the purpose of therapeutic intervention in atherosclerosis. Elucidating these mechanisms will not only advance our understanding of disease pathology, but also provide crucial insights into functional interactions that could be engineered to enhance lipid transporter expression as part of new therapeutic approaches to counteract atherosclerosis. The objective of this proposal is to determine how NCOA5, SND1, and SART1 function to control lipid transporter gene expression, function and foam cell formation. The central hypothesis is that these proteins are critical components of LXR transcriptional complexes assembled at the gene regulatory elements (GREs) of lipid transporters Abca1 and Abcg1, and that TLR-lipid crosstalk promotes changes in the composition and activity of these complexes, through phosphorylation, that favor gene repression, cholesterol accumulation and foam cell formation. Based on preliminary data, this hypothesis will be tested by pursuing three specific aims: (1) Elucidate the function of LXR-interacting proteins in the control of lipid transporter expression, function and foam cell formation; (2) Elucidate the role of phosphorylation of LXR-interacting proteins in the control of lipid transporter expression, function and foam cell formation; and (3) Characterize LXR-dependent regulatory complexes assembled on lipid transporter GREs during foam cell formation, and assess the role of NCOA5 in atherosclerosis, in vivo. Under the first aim, biochemical, molecular, and cellular approaches will be used to address the signal-dependent role of NCOA5, SND1, and SART1 in regulating expression of lipid transporters and foam cell formation. In the second aim, an innovative targeted mass spectrometry (MS) strategy will be used to identify signal-dependent changes in the phosphorylation state of NCOA5, SND1, and SART1, which will then be pursued using functional studies to define the impact of those modifications. For the third aim, an innovative promoter enrichment quantitative MS (PE-QMS) approach will be employed to characterize the composition of protein complexes assembled at GREs of Abca1 and Abcg1 in foam cells in vivo. Moreover, we will assess the importance of NCOA5 in promoting foam cell formation and atherosclerosis in vivo. The proposed research is significant because it will provide mechanisms to explain how foam cell formation is regulated at the molecular level, and provide insights into proteins and interactions that can be targeted to re-engineer gene regulatory networks to prevent foam cell formation and counteract atherosclerosis. This has the potential to benefit the health of patients suffering from this disease.